cAMP-mediated cell signaling regulates a myriad of important biological processes under both physiological and pathological conditions, including diabetes, heart failure, and cancer. In eukaryotic cells, the effects of cAMP are transduced by two intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (Epac/cAMP-GEF). Like PKA, Epac contains a cAMP-binding domain, an evolutionally conserved structural motif that acts as a molecular switch for sensing intracellular second messenger cAMP levels. The discovery of Epac has opened a new dimension in studying the cAMP-mediated signaling as both PKA and Epac are ubiquitously expressed in all tissues. An increase in intracellular cAMP levels will lead to the activation of both PKA and Epac. Therefore, the net cellular effects of cAMP are not just dictated by PKA or Epac alone, but dependent upon the dynamic expression of Epac and PKA and their specific subcellular distribution in a particular tissue. Currently, the physiological functions of Epac are not clear. One of the major challenges within the research field is the lack of Epac-specific antagonists to dissect the specific physiological functions that Epac play in the overall cAMP-mediated signaling. The objective of this proposal is to develop a High Throughput Screening (HTS) assay for the discovery of novel molecular probes that are specific for Epac but not PKA. These Epac-specific probes will be powerful pharmacological tools for investigating the biological functions of Epac and molecular mechanism of cAMP signaling as well as, for promoting an understanding of disease mechanisms related to Epac/cAMP signaling. PUBLIC HEALTH RELEVANCE: The goal of our proposed research is to develop a High Throughput Screening assay for the discovery of novel target-specific pharmacological probes that can be used for elucidating the mechanism of signal transduction mediated by an important second messenger, cAMP. The medical and pharmacological implications of this research program are far-reaching. Novel pharmacological probes targeting specific cAMP signaling components can potentially lead to the identification of mechanism-based therapeutic strategies for diseases associated with cAMP signaling.